ABSTRACT Many aspects of Zika virus (ZIKV) virology, immunology, and ecology are shared with other flaviviruses, particularly dengue virus (DENV). However, the 2015-2016 ZIKV epidemic in the Americas revealed that ZIKV can cause congenital infection and can spread through sexual contact, both unusual features of ZIKV compared to other flaviviruses. The immune mechanisms that protect against mosquito-borne flavivirus infection have been studied extensively, but since ZIKV is the first example of a sexually transmitted flavivirus, little is known about the immune mechanisms that protect against flavivirus sexual transmission. Furthermore, ZIKV infection in the female reproductive tract creates the possibility that ZIKV could access the fetal compartment via ascending transvaginal infection, in addition to the hematogenous transplacental route that occurs following mosquito-borne infection. The anatomic and immunologic barriers encountered in an ascending infection (mucosal immunity, cervix, amniotic membranes) are distinct from transplacental infection and understanding the potential for ZIKV to surmount these barriers is important for developing vaccines that will protect against ZIKV congenital infection. DENV and ZIKV share significant antigenic cross-reactivity, but the emergence of ZIKV in DENV-endemic areas provides strong epidemiological evidence that prior DENV infection does not provide durable protection against subsequent mosquito-borne ZIKV infection. We have shown that DENV antibodies bind to ZIKV virions but do not neutralize, generating antibody-opsonized viral particles that remain infectious. In vaginal mucus, even non- neutralizing antibodies can prevent infection, by trapping viral particles and preventing them from accessing vaginal epithelial cells. We will evaluate the antigenic specificity and neutralizing activity of antibodies in plasma and vaginal mucus from DENV- or ZIKV-immune women and determine whether ZIKV-binding IgG in human vaginal mucus can immobilize ZIKV virions. We will evaluate the contribution of non-neutralizing and neutralizing antibodies in vaginal mucus to protection from intravaginal ZIKV challenge in mice. Protection by cross-reactive DENV Abs would impact the protective correlates of an effective ZIKV vaccine, the epidemiology of ZIKV transmission in DENV-endemic areas, and the mechanisms of ZIKV congenital infection. Unlike other ascending vaginal pathogens, ZIKV also causes viremia, making it difficult to distinguish ascending and transplacental congenital infection. We will develop a new mouse model that uses neutralizing antibodies to restrict ZIKV infection to the female reproductive tract, allowing us to study ascending congenital infection specifically.